Brandi Wasek1, Erland Arning2, Teodoro Bottiglieri2. 1. Institute of Metabolic Disease, Baylor Scott and White Research Institute, Dallas, TX, United States. Electronic address: Brandi.WasekPatterson@BSWHealth.org. 2. Institute of Metabolic Disease, Baylor Scott and White Research Institute, Dallas, TX, United States.
Abstract
BACKGROUND: Assessing neurotransmitter metabolism in the brain is essential in studying the effects of drugs, dietary modification and characterizing transgenic mouse models of human neurodegenerative diseases. Regional brain concentrations of parent neurotransmitters and related metabolites are informative and provide a snap shot of the steady-state levels. The choice in method of sacrificing mice may differ from one laboratory to another, and the technique in removal of brain may have limitations depending on speed in which tissue can be dissected and frozen to prevent post-mortem changes. NEW METHODS: In order to better assess neurotransmitter metabolism in an effective and standardized manner we evaluated microwave irradiation as a method of sacrificing mice. Mice were sacrificed by CO2 asphyxiation followed by cervical dislocation or microwave irradiation at 4 Kw for 1.1 s. Brain tissue was harvested into five regions and stored at -80 °C until analysis by either LC-MS/MS for acetylcholine, choline and GABA, or HPLC-EC for dopamine, serotonin and norepinephrine and related metabolites. RESULTS: The results of our study showed considerable differences in the levels of neurotransmitters between the two methods of sacrifice. Overall, the concentrations of neurotransmitters were higher in mice sacrificed by microwave irradiation, except for GABA, which was lower. COMPARISON WITH EXISTING METHOD(S): Previous microwave irradiation studies employed presently outdated equipment and neurotransmitter analysis methods, and were not as comprehensive. CONCLUSIONS: The combination of microwave irradiation with LC-MS/MS and HPLC-EC detection allows accurate and sensitive measurement of several neurotransmitter systems in discrete mouse brain regions.
BACKGROUND: Assessing neurotransmitter metabolism in the brain is essential in studying the effects of drugs, dietary modification and characterizing transgenic mouse models of humanneurodegenerative diseases. Regional brain concentrations of parent neurotransmitters and related metabolites are informative and provide a snap shot of the steady-state levels. The choice in method of sacrificing mice may differ from one laboratory to another, and the technique in removal of brain may have limitations depending on speed in which tissue can be dissected and frozen to prevent post-mortem changes. NEW METHODS: In order to better assess neurotransmitter metabolism in an effective and standardized manner we evaluated microwave irradiation as a method of sacrificing mice. Mice were sacrificed by CO2 asphyxiation followed by cervical dislocation or microwave irradiation at 4 Kw for 1.1 s. Brain tissue was harvested into five regions and stored at -80 °C until analysis by either LC-MS/MS for acetylcholine, choline and GABA, or HPLC-EC for dopamine, serotonin and norepinephrine and related metabolites. RESULTS: The results of our study showed considerable differences in the levels of neurotransmitters between the two methods of sacrifice. Overall, the concentrations of neurotransmitters were higher in mice sacrificed by microwave irradiation, except for GABA, which was lower. COMPARISON WITH EXISTING METHOD(S): Previous microwave irradiation studies employed presently outdated equipment and neurotransmitter analysis methods, and were not as comprehensive. CONCLUSIONS: The combination of microwave irradiation with LC-MS/MS and HPLC-EC detection allows accurate and sensitive measurement of several neurotransmitter systems in discrete mouse brain regions.
Authors: Jens V Andersen; Niels H Skotte; Sofie K Christensen; Filip S Polli; Mohammad Shabani; Kia H Markussen; Henriette Haukedal; Emil W Westi; Marta Diaz-delCastillo; Ramon C Sun; Kristi A Kohlmeier; Arne Schousboe; Matthew S Gentry; Heikki Tanila; Kristine K Freude; Blanca I Aldana; Matthias Mann; Helle S Waagepetersen Journal: Cell Death Dis Date: 2021-10-16 Impact factor: 8.469